The Fundamental Research And Application Of Mixed-mode High Performance Liquid Chromatography For Protein Purification

High performance liquid chromatography(HPLC) is widely used in various fields for high-resolution, fast-separation and well-repeatability. However, due to the similar properties of proteins, the appllicaiotn of HPLC in separation of macromolecular was restricted by the single mode function, and this HPLC can not satisfy the purification of high purity of proteins. Hence, several mixed-mode chromatography(MMC) has been developed in recent years, such as Hydrophobic charge induction chromatography(HCIC). HCIC is a kind of mixed mode chromatography with multiform functions, commonly combining thiophilic, hydrophobic, and electrostatic interactions. The target protein can be adsorbed on the HCIC ligand by thiophilic and hydrophobic effects and desorbed under the electrostatic repulsion between protein and the charged groups. In this work, the HCIC was introduced to HPLC, forming a new kind of High Performance Hydrophobic Charge Induction Chromatography(HPHCIC), and the HPHCIC was explored for protein separation and analysis. The main work were listed as follow:(1) Preparation of HPHCIC resins: Three kinds of HPHCIC resins were prepared by coupling 2-mercapto-1-methyl imidazole, 3-aminopyridine and 2-mercaptobenzimidazole onto the silica gel(particle size 5 μm, aperture 300 ?) via KH560 as the activator. The result indicated that the highest KH560 activation density is 165μmol?g-1 at the condition of 90℃ and 6 h; the density of ligands on resin were 105, 85 and 45 μmol?g-1 for 2-mercapto 1-methyl imidazole, 3-aminopyridine and 2-mercaptobenzimidazole, respectively, with the condition of 40℃ and 6 h.(2) Retention behavior of proteins in HPHCIC: BSA, IgG and Lysozyme were used as the model proteins for investigation of retention behavior in HPHCIC, with a column endcapped with trimethyl chlorosilane as the blank control. The effect of pH, salt density and acetonitrile content in mobile phase on the retention factor of three proteins in HPHCIC were investigated. The results show a certain regularity for the retention behavior which affected by the hydrophobic and electrostatic properties of proteins and ligands, and the condition of mobile phase. The repulsive force could be recognized as: 3-aminopyridine> 2-mercapto-1-methyl imidazole>2-mercaptobenzimidazole, while the interation were wholely on the contrary. The ligands of 2-mercapto-1-methyl imidazole could be considered as the potential ligand for HPHCIC due to its appropriate hydrophobic and electrostatic interactions.(3) Separation of model protein using HPHCIC column: The effect of different mobile phase pH condition on the separation behavior of three proteins in HPHCIC were investigated. It is found that the resolution of proteins on column of 3-aminopyridine is low while the BSA and IgG are almost adsorpted on column of 2-mercaptobenzimidazole. The resolution of proteins on column of 2-mercapto-1-methyl imidazole are relatively higher than that on other two columns, and the peak of protein on column of 2-mercapto-1-methyl imidazole is much better. Hence, the ligand of 2-mercapto-1-methyl imidazole was selected for further investigation of separation of mix model proteins. The results indicates that the acetonitrile content in mobile phase with low pH has the greatest impact on the resolution, while the gradient of salt and pH are not important. The separation behavior of three proteins in HPHCIC gives a guide for the application of HPHCIC with 2-mercapto-1-methyl imidazole as ligand.(4) Application of HPHCIC in protein separation and analysis: 1. First, the recombination Protein A was expressed by E.Coli BL21. Then the cell was disrupted and the supernate was collected by centrifugation. After pretreatment, Protein A was analyzed using HPHCIC. The results show that the peak of Protein A is clear and the resolution is acceptable which compared with the column of C18. 2. Lysozyme in egg white solution was also analyzed using HPHCIC. Similar to the separation of Protein A, the resolution of lysozyme is also good and the result was proved by comparation with high performance reversed phase chromatography.